Crosslinked, porous, polyacrylate beads

ABSTRACT

Uniformly-shaped, porous, round beads are prepared by the co-polymerization of an acrylic monomer and a cross-linking agent in the presence of 0.05 to 5% by weight of an aqueous soluble polymer such as polyethylene oxide. Cross-linking proceeds at high temperature above about 50° C or at a lower temperature with irradiation. Beads of even shape and even size distribution of less than 2 micron diameter are formed. The beads will find use as adsorbents in chromatography and as markers for studies of cell surface receptors.

ORIGIN OF THE INVENTION

The invention described herein was made in the performance of work undera NASA contract and is subject to the provisions of Section 305 of theNational Aeronautics and Space Act of 1958, Public Law 83-568 (72 Stat.435; 42 USC 2457).

This is a division, of application Ser. No. 434,124, filed Jan. 17, 1974now U.S. Pat. No. 3,957,741.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to uniformly-sized, porous, small, round,hydrophilic polymeric beads and methods of making the beads and to theuse of the beads in separation of molecules and in various analyticaland diagnostic techniques.

The invention is directed to particles and the methods by which they aremade and does not include tracer tagging or biological and clinicalapplication tagging aspects disclosed and claimed in copending U.S.patent aplication Ser. No. 177,017, filed Sept. 1, 1971, by William J.Dreyer.

2. Description of the Prior Art

There is a need for small, stable, spherical particles which arebio-compatible, i.e., do not interact non-specifically with cells orother biological components and which contain functional groups to whichspecific proteins and other bio-chemical molecules can be covalentlybonded by established chemical procedures. Poly HEMA, apolyhydroxyethylmethacrylate, also known under the name Hydrogel, hasbeen shown to possess blood compatible properties and is also used inthe manufacture of contact lenses. The hydroxyl groups can be activatedby cyanogen bromide for covalent bonding of proteins and other chemicalscontaining amino groups to the polymeric latex. Methacrylic acidresidues which impart a negative charge onto the particles tend toprevent non-specific binding to cell surfaces and to provide carboxylgroups to which a variety of bio-chemical molecules can be covalentlybonded using the carbodiimide method. Cross-linking of the polymericmatrix is essential to maintain the stability and size of the particlesin both aqueous solution and in organic solvents commonly used in thefixation and dehydration of biological specimens for light and electronmicroscopy.

Knowledge of the nature, number and distribution of specific receptorson cell surfaces is of central importance for an understanding of themolecular basis underlying such biological phenomena as cell-cellrecognition in development, cell communication and regulation byhormones and chemical transmitters, and differences in normal and tumorcell surfaces. In previous studies, the localization of antigens andcarbohydrate residues on the surface of cells, notably red blood cellsand lymphocytes, has been determined by bonding antibodies or lectins tosuch macromolecules as ferritin, hemocyanin or peroxidase which haveserved as markers for transmission electron microscopy. With advances inhigh resolution scanning electron microscopy (SEM), however, thetopographical distribution of molecular receptors on the surfaces ofcell and tissue specimens can be readily determined by similarhistochemical techniques using newly developed markers resolvable bySEM.

Recently commercially available polystyrene latex particles have beenutilized as immunologic markers for use in the SEM technique. Thesurface of such polystyrene particles is hydrophobic and hence certaintypes of macromolecules such as antibodies are adsorbed on the surfaceunder carefully controlled conditions. However, such particles sticknon-specifically to many surfaces and molecules and this seriouslylimits their broad application. These particles are uncharged and arenot capable of any ionic or covalent bonding of protein and otherbiological molecules.

HEMA particles possess chemical groups suitable for covalent bonding.However, homopolymers of HEMA are generally too soft for formation ofporous beads and conventional suspension polymerization techniques arefound to form fairly large particles on the order of 40 to 60 microns.Red blood cells and lymphocytes have a size of the order of 8 to 10microns and in order to bind to specific receptor sites the beads mustbe of an order of magnitude smaller than the biological cell.

Small, uniformly-sized, cross-linked, porous, polyacrylic beads willalso find use as a low-cost, stable adsorbent in separating andpurifying organic and inorganic compounds including polymers. The beadswill also find use in chromatographic separation, filtration and gelpermeation and affinity chromatography.

The hydrophilic organic gels commonly used in chromatography aresparsely cross-linked xerogels with a high swellability capacity in theeluent. They are characterized by considerable capacity ratio. However,the mechanical strength of the particles in the swollen state rapidlydecreases with decreasing density of the cross-links. Application of theeluent under pressure at the column inlet frequency leads to plugging.Therefore, these gels are not suitable for high-speed gelchromatography. A new hydrophilic packing for gel chromatography isneeded exhibiting high mechanical and hydrolytic stability.

SUMMARY OF THE INVENTION

In accordance with the invention, small, round, porous beads ofuniformly small diameter are provided. The beads are formed from anacrylic monomer containing a hydroxy group. The beads are hydrolyticallystable and of sufficient mechanical strength to be useful as anadsorbent in separating and purifying organic and inorganic compoundsand will find use in column or in film chromatography, gel filtrationand permeation, separation and analysis. The beads are of wellcharacterized structure and of outstanding purity.

Small spheres covalently bonded to antibodies and other biologicalmaterials are useful as specific cell surface markers for scanningelectron microscopy. The particles are found to bind to hormones,toxins, lectins and other molecules and have application in thedetection and localization of a variety of cell surface receptors.Particles tagged with fluorescent dye or radioactive molecules serve assensitive markers for fluorescent microscopy and as reagents forquantitative study of cell surface components by covalently bondinglectins, antigens, hormones and other molecules to these spheres, thedetection and localization of specific carbohydrate residues,antibodies, hormone receptors and other specific cell surface componentscan also be determined. These reagents also have applications in highlysensitive radioimmune assays as visual markers for light, fluorescentand transmission electron microscopy, for radioactive quantitation ofspecific cell surface receptors and as potential therapeutic reagents.

Uniformly-shaped, porous, round beads are prepared by co-polymerizationof an acrylic monomer and a cross-linking agent in the presence of 0.1to 5%, preferably 0.2 to 2%, by weight of an aqueous soluble polymersuch as polyethylene oxide. Cross-linking proceeds at high temperatureabove about 50° C. or at a lower temperature with irradiation. Beads ofeven shape and even size distribution of less than 2 micron diameter areformed. The beads will find use as adsorbents in chromatography and asmarkers for studies of cell surface receptors.

These and other objects and many attendant advantages of the inventionwill become apparent as the description proceeds.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The beads are prepared by the aqueous suspension polymerization of amonounsaturated, hydroxy substituted, liquid acrylic monomer and across-linking agent in the presence of 0.1 to 5% of a water solublepolymeric suspending agent. Polymerization proceeds at a temperatureabove about 50° C., preferably 70° C. to reflux in the presence orabsence of a catalyst or at a lower temperature of -70° C. to 70° C.with application of high energy radiation to the polymerizable mixture.

The monomer is suitably a hydroxy alkyl substituted acrylate oracrylamide or an amino alkyl substituted acrylate. Representativemonomers may be selected from compounds of the formula: ##STR1## whereR¹ is hydrogen or lower alkyl of 1-8 carbon atoms, R² is alkylene of1-12 carbon atoms, Z is OH or ##STR2## where R³ or R⁴ are H, lower alkylor lower alkoxy. Hydroxyethylmethacrylate, hydroxypropyl methacrylate,dimethylaminoethyl methacrylate and 2-aminoethyl methacrylate arereadily available commercially. Minor amounts of 1-35%, suitably 10-25%,of a compatible comonomer such as a lower alkyl methacrylate, acrylic ormethacrylic acid, styrene or vinyl toluene may be present in thepolymerizable mixture.

The cross-linking agent is present in the polymerizable mixture in anamount from 0.1 to 30% and is a liquid polyunsaturated compound such asa diene or a triene capable of addition polymerization with theunsaturated group of the monomer. Suitable compounds are low molecularweight liquid polyvinyl compounds such as ethylene dimethacrylate,divinyl benzene, trimethylol propane trimethacrylate andN,N'-methylene-bis-acrylamide.

A commercial form (94%) of hydroxyethylmethacrylate (HEMA) andhydroxypropyl methacrylate (HPMA) as supplied, contains small amounts ofmethacrylic acid, hydroxyalkoxyalkylmethacrylate and dimethacrylates -ethylene dimethacrylate in HEMA and propylene dimethacrylate in HPMA.HPMA is generally a mixture in which the principal monomers comprise68-75% of 2-hydroxypropyl and 25-32% of1-methyl-2-hydroxyethylmethacrylate. Typical compositions in weightpercentage follow:

    ______________________________________                                        Compound         HEMA - 94%  HPMA - 94%                                       ______________________________________                                        Hydroxyalkylmethacrylate                                                                       86          87                                               Higher boiling methacrylate,                                                  Principally hydroxyalkoxy-                                                    alklymethacrylate                                                                              6           5                                                Methacrylic Acid 3.5         4.5                                              Dimethacrylate   1.5         0.7                                              ______________________________________                                    

The monomers are diluted in aqueous medium at a level of from 5 to 50%by weight. The aqueous medium comprises water and the water solublepolymer. The water soluble polymer may be present in an amount as low as0.05 weight percent. Amounts above 5% are believed unnecessary andrequire added time and effort to remove the polymer from the finalbeads.

Finely and uniformly shaped and sized beads have consistently beenproduced in an aqueous medium containing a polyether. The polyethersgenerally have a molecular weight from 300,000 to 10,000,000, preferably400,000 to 6,000,000, and are polymers of alkylene oxides such asethylene oxide, propylene oxide or their mixtures. Polyethylene oxidesare preferred due to their solubility in water.

The polymerization proceeds without catalyst and without stirring withapplication of heat to the mixture at a temperature of from 70° C. toreflux, generally about 100° C. or with application of high energyradiation capable of generating free radicals and initiatingpolymerization and forming cross-linking bonds between olefinic groups.Polymerization proceeds by application of 0.05 to 1.0 megarads ofradiation from a cobalt gamma source at a temperature of 0° C. to 70° C.The reaction is preferably conducted under oxygen excluding conditions,generally by applying vacuum to the reaction vessel or by displacing thehead space with an inert gas such as nitrogen. A free radical catalystsuch as ammonium persulfate and additional agents such as othersuspending or emulsifying agents may be present in the polymerizablemixture.

After polymerization has proceeded to completion, the polymerizationmixture is diluted with hot water and filtered and washed with boilingwater to remove the polyether. The dry material in over 90% yield is inthe form of separate round beads or agglomerates of beads. Agglomerates,if present, are subdivided into beads mechanically by dispersion in anon-solvent liquid, crushing or grinding. The beads are uniformly sizedand at least 80% and preferably at least 90% of the beads are of auniform diameter less than 5 microns, preferably from 0.001 to 2microns. The cross-linked porous beads are insoluble and swellable inwater and are insoluble in common inorganic and organic solvents.

Specific examples of practice follow.

EXAMPLE 1

Commercial 94% HEMA containing 1.5 weight percent ethylenedimethacrylate and 0.5% hydroquinone as a stabilizer was vacuumdistilled at 97°-99° C at a pressure of 1 mm Hg. 14 g of the freshlydistilled HEMA was dissolved in 180 g of water. 4 g of a polyethyleneoxide polymer of a molecular weight of about 4,000,000 was added and themixture heated at mild reflux (98° C. ± 2° C.) for 24 hours. Thepolymerized material was then diluted with hot water and filteredthrough a wire mesh. The separated solid on the wire mesh was washedwith boiling water until the polyethylene oxide was removed. The drymaterial in a yield of over 90% was ground into individual beads, 80% ofwhich had a diameter of 2 microns.

EXAMPLE 2

A mixture of 200 g of freshly vacuum distilled HEMA, 4 g of 1 × 10⁶molecular weight polyethylene oxide and 30 g of trimethylol propanetrimethacrylate was diluted to 1 liter with water and nitrogen inerted.0.1 Megarad of radiation was applied to the mixture at room temperaturefrom a cobalt gamma source over a period of about 15 minutes. The beadswere filtered, washed with boiling water several times and centrifugedto provide a 99% yield.

Under scanning electron microscope the dry beads were determined to beround and at least 90% were of a 1.6 micron diameter.

EXAMPLE 3

The following aqueous mixture was prepared.

    ______________________________________                                        Component                 Weight %                                            ______________________________________                                        HEMA (Freshly distilled containing                                            1.5 % ethylene dimethacrylate)                                                                          16                                                  Trimethylol propane trimethacrylate (TPT)                                                               0.6                                                 Polyethylene oxide (M.W. 10.sup.6)                                                                      0.4                                                 Dimethylaminoethyl methacrylate                                                                         4.0                                                 ______________________________________                                    

The mixture was nitrogen inerted and 0.1 megarads of radiation wasapplied to the mixture at room temperature from a cobalt gamma sourcefor 15 minutes. The beads were recovered and separated as in Example 2in 99% yield. Under scanning electron microscope, the diameter of over90% of the beads was determined to be from 1-2 microns. The copolymerbeads contain hydroxyl as well as dimethylamino groups. The procedurewas repeated at 0° C. in ice bath with 0.2 megarads over a period of 30minutes with the same results.

EXAMPLE 4

The following aqueous mixture was prepared.

    ______________________________________                                                Component      Weight %                                               ______________________________________                                        Acrylamide             20.6                                                   N,N'-methylene-bis-acrylamide                                                                        0.6                                                    Polyethylene oxide (M.W. 10.sup.6)                                                                   0.4                                                    ______________________________________                                    

The mixture was polymerized under the conditions of Example 3 to yieldagglomerated beads which were subdivided into individual beads having adiameter from 1-2 microns.

EXAMPLE 5

The following aqueous mixture was prepared.

    ______________________________________                                                Component      Weight %                                               ______________________________________                                        Dimethylaminoethyl methacrylate                                                                      20.6                                                   TPT                    0.6                                                    Polyethylene oxide (M.W. 10.sup.6)                                                                   0.4                                                    ______________________________________                                    

The mixture was nitrogen inerted and subjected to 0.1 megarad of cobaltgamma radiation for 15 minutes. Individual beads of 1-2 micron diameterwere produced.

The small, pure, round, uniformly-sized beads of this invention can beutilized for the labeling of biological cells such as lymphocytes. Adiseased condition can be diagnosed by binding an antibody to beads,mixing the beads with a body serum and observing whether the beads bindto specific antigen sites causing precipitation or agglutination. Thepresence of OH, COOH and amine groups on the beads permits covalentbonding of biomolecules such as haptens, enzymes, antibodies or lectinsto the beads by means of cyanogen bromide, carbodiimide orglutaraldehyde reactions. Diagnosible conditions are hepatitis,gonorrhea, rheumatoid arthritis, streptococcus infections, andpregnancy. Labeled beads may also be used for blood typing.

The beads also bind fluorescent dyes and will find use in fluorescentmicroscopy. Since the dye is bound to the bead particle and not to theantibody, a high degree of tagging can be attained without adverselyaffecting the antibody activity for studies requiring high sensitivity.The fluorescent dye bound beads can also act as a marker for cells byadding the beads to cells, in vitro or in vivo, and the attachment tospecific cells observed.

EXAMPLE 6

0.2 g of the beads of Example 2 were suspended in 20 ml of H₂ O,homogenized in a glass homogenizer and transferred to a beaker. 0.8 g ofcyanogen bromide was added while stirring. The pH was maintained at10-11 by addition of 2N NaOH. 5 mg of epsilon dansyl lysine was addedand stirring continued for 1 hour. The mixture was centrifuged 6 timeswith distilled water. The supernatant liquid was not fluorescent afterthe sixth centrifugation. Highly fluorescent beads were recovered asdetermined by fluorescent microscopy.

EXAMPLE 7

0.1 g of 9-amino acridine hydrochloride were bound to 0.2 g of the beadsof Example 2 following the procedure of Example 6. The beads were highlyfluorescent.

Due to the hydrophilic and purity characteristics of the porous, round,finely-sized beads, the beads will also find use in affinitychromatography, column or thin film chromatography, gel filtration orpermeation. The beads are capable of purification of highly chargedsynthetic and natural polyelectrolytes and can be used to separate bothorganic and inorganic molecules.

It is to be realized that only preferred embodiments of the inventionhave been described, and that numerous substitutions, alterations andmodifications are all permissible without departing from the spirit andscope of the invention as defined in the following claims.

What is claimed is:
 1. A composition consisting essentially of uniformlyshaped, hydrophilic, swellable, porous, round beads, at least 80% havinga diameter less than 2 microns and being the cross-linked polymer ofacrylamide and 0.1 to 30 weight percent of a liquid diene or trienecross-linking agent.
 2. A composition according to claim 1 in which atleast 90% of the beads have a uniform diameter between 0.001 and 2microns.
 3. A composition according to claim 1 in which thecross-linking agent is N,N'-methylene-bis-acrylamide.
 4. A compositionaccording to claim 1 in which the beads contain 1-35 weight percentbased on acrylamide of a comonomer selected from a lower alkylmethacrylate, acrylic acid, methacrylic acid, styrene or vinyl toluene.